The hematopoietic stem cell (HSC) is the best-studied stem cell type in adults and it is also the only stem cell type with proof of clinical success. However, the biggest challenge for a broader use of HSCs remains the true expansion of the stem cells in vitro. While HSCs may divide in response to certain cytokine combinations in vitro, they tend to differentiate and lose their self-renewal potential after their divisions. Therefore, understanding the intrinsic mechanisms underlying the self-renewing division of a HSC is one of the central issues that directly determine our ability to manipulate HSCs for clinical applications. The cell cycle is a key process by which the fate of HSCs may be independently regulated. Our studies, as well as others, have demonstrated that hematopoietic cell proliferation is largely maintained in a differentiation- dependent manner by cell cycle inhibitors, namely the cyclin-dependent kinase inhibitors (CKIs). Especially, we have recently reported a striking and distinct role of the CKI, p18INK4c (p18), in HSCs as evidenced by the significantly enhanced long-term engraftment potential of HSCs in the absence of p18. Interestingly, our preliminary data indicate that absence of p18 does not cause a general increase of cell proliferation in most hematopoietic cell types in vivo. Based on these results, we hypothesize that p18 may serve as a specific inhibitor for the self-renewing process of HSCs and that targeting p18 in HSCs may enhance HSC expansion/function in vitro. In the current proposal, we plan to obtain direct evidence for the involvement of p18 in the self-renewing division of a HSC at the single cell level. To further define the potential link of p18 and other CKIs with some known self-renewal pathways, mainly the Notch pathway, we will also document the expression levels of p18 in response to activation of these upstream signaling pathways in conjunction with other functional assessments in murine models. Finally, a similar effect will be explored in human HSCs using the RNA interference technology in a pre-clinical setting. The proposed studies will set the stage for our appreciation of the cell cycle interface during the self-renewing process of HSCs and therefore offer potential new strategies for manipulating human HSCs and possibly other adult stem cell populations as well.